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Plant Physiol. (1999) 120: 23-32
Messenger RNA-Binding Properties of Nonpolysomal
Ribonucleoproteins from Heat-Stressed Tomato Cells1
Rogier Stuger2,
Sigrid Ranostaj,
Tilo Materna3, and
Christoph Forreiter*
Department of Molecular Cell Biology, Goethe University, Marie
Curie Strasse 9, 60439 Frankfurt am Main, Germany
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ABSTRACT |
Most
cells experiencing heat stress reprogram their translational machinery
to favor the synthesis of heat-stress proteins. Translation of other
transcripts is almost completely repressed, but most untranslated
messengers are not degraded. In contrast to yeast, Drosophila
melanogaster, and HeLa cells, plant cells store repressed
messengers in cytoplasmic nonpolysomal ribonucleoproteins (RNPs). To
follow the fate of untranslated transcripts, we studied protein
composition, mRNA content, and RNA-binding properties of nonpolysomal
RNPs from heat-stressed tomato (Lycopersicon peruvianum) cells. Contrary to the selective interaction in vivo, RNPs isolated from tomato cells bound both stress-induced and repressed messengers, suggesting that the selection mechanism resides elsewhere. This binding
was independent of a cap or a poly(A) tail. The possible role of
proteasomes and heat-stress granules (HSGs) in mRNA storage is a topic
of debate. We found in vitro messenger-RNA-binding activity in
messenger RNP fractions free of C2-subunit-containing proteasomes and
HSGs. In addition, mRNAs introduced into tobacco (Nicotiana
plumbaginifolia) protoplasts were found in the cytoplasm but were not associated with HSGs.
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INTRODUCTION |
Cells in stress alter their activities to minimize damage and
facilitate recovery. The heat-stress response in eukaryotes involves
remarkable reprogramming of translation: Most mRNAs are repressed, but
those encoding HSPs are efficiently translated (Lindquist, 1986 ; Nover,
1991; Sierra and Zapata, 1994; Brostrom and Brostrom, 1998). Although
translational reprogramming occurs in a wide range of cells, it is
achieved in different ways (Sierra and Zapata, 1994). Yeast translates
HSP and non-HSP messengers at the onset of stress (Bienz, 1982;
Lindquist et al., 1982); preferential synthesis of HSPs through
cytoplasmic enrichment of the corresponding messengers results from a
continuous supply of HSP mRNAs, nuclear retention of other messengers
(Saavedra et al., 1997), and rapid turnover of cytoplasmic transcripts. Drosophila melanogaster and HeLa cells maintain non-HSP
mRNAs on polysomes, but their translation is dramatically reduced
(Ballinger and Pardue, 1982; Hickey and Weber, 1982; Lindquist et al.,
1982). Plants control translation during heat stress in a different
way. Most repressed messengers dissociate from polysomes and are
transiently stored elsewhere in the cytoplasm (Nover et al., 1989;
Apuya and Zimmerman, 1992). The mechanism of this process and the
proteins involved are still not known, although several models for mRNA storage have been proposed.
The appearance of large RNA-containing particles (Neumann et al., 1984)
and structures called HSGs (which are rich in small HSPs; Nover et al.,
1983) in heat-stressed tomato (Lycopersicon peruvianum)
cells led to the hypothesis that mRNA is stored in these structures
(Nover et al., 1989). This implies a novel role for small HSPs in
addition to their function as molecular chaperones (Jakob et al., 1993;
Lee et al., 1995; Forreiter et al., 1997). Indeed, cytoplasmic
fractions enriched in small HSPs contain mRNAs that can be translated
in vitro into proteins similar to those encoded by polysomal messengers
from nonstressed cells, whereas in vitro translation of polysomal mRNAs
from stressed cells mainly yields HSPs (Nover et al., 1989).
Translation is reprogrammed immediately upon heat stress, whereas HSGs
appear only after prolonged stress. In addition, Gallie and Pitto
(1996) reported that translational switch and increased transcript
stability occur when small HSP synthesis is blocked. Alternatively,
messengers may be stored in RNA and protease-containing particles
called prosomes or proteasomes (Scherrer and Bey, 1994; Schmid et al.,
1995). Their RNA content seems inversely related to the purity of the
preparation, and the inhibition of translation by proteasomes in vitro
that is not caused by proteolysis may be due to proteasome-associated
RNase activity (Schmid et al., 1995). This activity is incompatible
with RNA storage and stabilization. Other sites of storage cannot be
ruled out, such as a plant homolog of a translation- regulating
particle identified in animal cells that is rich in the 72-kD
poly(A)-binding protein, and mRNA-binding proteins of 50 to 60 kD
termed mRNP core proteins (Evdokimova et al., 1995; Spirin, 1996;
Yurkova and Murray, 1997). However, plant counterparts of core mRNPs
have not yet been found, so storage of repressed mRNA in plants during
stress remains enigmatic.
In this study we analyzed the partitioning of stress-induced and
repressed transcripts over polysomes and npRNPs from tomato cells. Messengers repressed by heat stress were found in nonpolysomal storage particles and reappeared on polysomes during recovery. However,
stress-induced and repressed mRNAs could bind isolated npRNPs in vitro,
suggesting that the selection mechanism resided elsewhere. Storage
particles bound messengers in an RNase-resistant form in the absence of
small HSPs and C2-subunit-containing proteasomes. In addition, mRNAs
introduced into tobacco (Nicotiana plumbaginifolia) cells
did not colocalize with small HSPs.
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MATERIALS AND METHODS |
Tomato Cell Culture and Heat Stress
The tomato (Lycopersicon peruvianum LpVII)
cell-suspension culture was maintained as described previously
(Nover et al., 1982). Heat treatments of exponentially growing cells
were 25°C (control), or 15 min at 40°C followed by 3 h at 25°C
(pre-induced). Stressed cells were treated for 15 min at 40°C then
subsequently incubated 3 h at 25°C with an additional heat treatment
of 2 h at 40°C. Recovered cells were obtained after the full stress
treatment with a subsequent incubation of 2 h at 25°C.
Cell Fractionation
Isolation of npRNPs was modified from Nover et al. (1983, 1989).
Samples were kept on ice and centrifuged at 4°C. Cells were harvested
by aspiration, ground in liquid nitrogen, and placed in 50 mM Tris-HCl, pH 7.8, 0.5 mM
MgCl2, 25 mM KCl, 2 mM
CaCl2, 1 mM EDTA, 0.1% Nonidet P-40,
0.1%  -mercaptoethanol, 25% glycerol, and 150 mM Suc at
2 mL g 1 fresh weight of cells. Debris was
removed by 5 min of centrifugation at 3,400g. Polysomes were
disrupted by adding 250 mM
KCl, 30 mM EDTA, and 0.5%
Nonidet P-40. After 20 min of centrifugation at 6,700g, the
supernatant was centrifuged for 1 h at 68,000g through two Suc pads: 5 mL of 15% Suc, 1 mM EDTA, and 10 mM MgCl2 in a gradient
buffer (20 mM Tris-HCl, pH 7.8, 250 mM KCl, 25% glycerol, and 0.1%
-mercaptoethanol) overlaid with 5 mL of 10% Suc and 30 mM EDTA in a gradient buffer. Supernatants (S30)
were used to prepare P100 fractions (see below). Pellets were
homogenized in 10 mL of 20 mM Tris-HCl, pH 7.8, 250 mM NaCl, 30 mM EDTA,
0.5% Nonidet P-40, 0.2% lauroylsarcosine, 0.1% -mercaptoethanol,
and 15% glycerol with a Teflon homogenizer and spun for 5 min at 5,000 rpm.
The resulting supernatants were centrifuged as above through 15 mL each
of 15% and 10% Suc solutions. Resedimented P30 pellets and P100
fractions, prepared by spinning S30s through a 2 mL 15% Suc pad for
16 h at 220,000g, were rinsed twice with RNP buffer (20 mM Tris-HCl, pH 7.8, 50 mM
NaCl, 0.1% -mercaptoethanol, and 15% glycerol) and resuspended in
the same buffer. To isolate polysomes, 25 µg
mL1 cycloheximide
was added to cell cultures 1 min before harvesting. Cells were ground
in liquid nitrogen and thawed in PIB buffer (100 mM Hepes-KOH, pH 8.3, 500 mM KCl, 20 mM
MgCl2, 2 mM EDTA, 0.5%
Triton X-100, 0.05% -mercaptoethanol, and 250 mM Suc). Debris was removed by spinning for 10 min at 8,000g. After centrifugation for 3 h at
150,000g through pads of 20% and 60% Suc in PIB buffer, polysomes were collected from the borders of the pads.
RNA-Blot Analysis
Antisense probes were synthesized with an RNA-labeling kit
(Boehringer Mannheim) from templates encoding tomato histone H4, calmodulin, and cyclophilin (Materna, 1996), and from plasmids encoding steroid dehydrogenase (Ganal et al., 1998), HsfA2 (Scharf et
al., 1990), and HSP17.7 (GenBank accession no. AJ225046). RNA was
isolated from cells, polysomes, and P30s using guanidinum isothiocyanate (Forreiter and Apel, 1993) and separated on 1.2% agarose gels containing 1% formaldehyde. It was then transferred to
Hybond N+ membranes (Amersham) and fixed
using a Stratalinker (Stratagene). Blots were prehybridized for 5 min
in 5× SSC, 50% deionized formamide, 0.02% SDS, 0.1%
lauroylsarcosine, and 2% Denhardt's reagent; they were hybridized in
this solution with DIG-labeled probes for 16 h at 65°C and given
three 10-min washes at 62°C with 0.1× SSC and 0.1% SDS. For
detection we used anti- DIG-alkaline phosphatase (Boehringer Mannheim),
nitroblue tetrazolium salt, and 5-bromo-4-chloro-3-indolyl phosphate,
following the manufacturer's instructions.
mRNA Synthesis
Tomato histone H4 (GenBank accession no. X69179) and HSP17.7 cDNAs
amplified by PCR were inserted into pBluescript SK (+) (Stratagene).
The HSP17.7 construct was linearized with BamHI. A linear
template containing T7 and T3 promoters was amplified from the histone
clone with primers M13( 20) and M13 reverse (Stratagene). The
luciferase mRNAs luc and lucA50 were produced
from pT7-LUC-A50 (Gallie et al., 1991) cut with BamHI and
DraI, respectively. Capped and uncapped RNAs were
transcribed with the mCap kit (Stratagene) and RNase inhibitor (RNasin,
Promega), [ -32P]UTP (New England Nuclear),
or fluorescin-12-UTP (Boehringer Mannheim). DNA removal by DNase I
(Stratagene) was followed by spinning through Sephadex G50 and ethanol
precipitation as described by Sambrook et al. (1989).
RNA Protection Assay
Fluorescin-labeled mRNA (approximately 200 ng) and 40 µg of
npRNPs (or 50 µg of BSA) in RNP buffer were incubated on ice for 10 min in the presence of 20 units of RNasin (Promega), which does not
inhibit micrococcal nuclease. After treatment with 2 units of
micrococcal nuclease (Boehringer Mannheim) and 5 mM
CaCl2 for 10 min at 37°C, EDTA was added to 20 mM. RNAs were separated on 7 M urea/6%
polyacrylamide gels (Sambrook et al., 1989).
Gel Retardation Assay
Radioactive mRNA (105 cpm) and 10 µg of
npRNPs (or 50 µg of BSA) in RNP buffer were incubated on ice for 10 min, mixed with 5× loading buffer (50% glycerol, 5× TBE, 50 mM EDTA, and bromphenol blue), and separated on a 5%
polyacrylamide/10% glycerol gel in TBE at 4°C.
Gel Filtration Chromatography
RNPs from 0.5 g fresh weight of tomato cells were incubated
with 105 cpm 32P-labeled
mRNA for 10 min on ice, applied to a Superdex S200 (Pharmacia) column,
and eluted with 20 mM Tris-HCl, pH 7.8, at 1 mL
min1 using a Gradifrac (Pharmacia).
Radioactivity in 10-mL fractions was measured in a scintillation
counter, and proteins were precipitated with acetone and analyzed by
SDS-PAGE.
Protein Analysis
Proteins separated on 15% SDS-PAGE gels (Laemmli, 1970) were
stained with Coomassie blue or blotted onto Hybond C nitrocellulose (Amersham). Blots were blocked with 5% nonfat dry milk in TBS, probed
with antibodies against HSP17 (obtained from M. Kirschner, Frankfurt,
Germany), HSP70 (StressGen, Victoria, Canada), HsfA2 (Lyck et
al., 1997), tubulin (Sigma), or the proteasome C2 subunit (Umeda
et al., 1997), and developed with anti-rabbit horseradish peroxidase
(Bio-Rad) and enhanced chemiluminescence (DuPont-New England Nuclear )
or with anti-rabbit alkaline phosphatase (Promega), nitroblue
tetrazolium, and 5-bromo-4-chloro-3-indolyl phosphate, following the
manufacturer's instructions.
Localization of mRNA in Tobacco Protoplasts
Tobacco (Nicotiana plumbaginifolia) leaf protoplasts
were prepared according to the method of Treuter et al. (1993) and then washed five times to remove RNases from the cell wall-digesting enzyme
solution. Fluorescein-labeled RNA was introduced using PEG as described
for DNA by Treuter et al. (1993). After transformation, the PEG
concentration was reduced by adding 10 volumes of culture medium.
Control cells were kept at 25°C for 2 h following
transformation. Pre-induced cells (15 min at 40°C and 2.5 h at
25°C) were transformed and stressed by gradual heating to 37°C over
15 min and subsequent incubation at this temperature for 105 min. Cells
were fixed with 3% paraformaldehyde for 30 min, incubated for 15 min
in 1% Nonidet P-40/PBS, and attached to polylysine-coated coverslips.
After 10 min of incubation in 2% Nonidet P-40/PBS, two 10-min washes with PBS, and 30 min of blocking with 1% BSA/PBS, cells were incubated overnight at 4°C with anti-HSP17, washed three times, reblocked, and
incubated for 2 h at 37°C with anti rabbit-tetramethyrhodamine isothiocyanate (Sigma). After three washes, cells were mounted in PBS (75% glycerol and 0.1% phenylenediamine) and analyzed by confocal fluorescence microscopy.
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RESULTS |
Protein Composition of Nonpolysomal mRNP Fractions from Tomato
Cells
Cytoplasmic fractions enriched in RNPs were sedimented from tomato
cells by ultracentrifugation, yielding P30 fractions, and by prolonged
ultracentrifugation of the P30 supernatants, yielding P100s (Fig.
1A). P30s from heat- stressed cells have
been reported to contain HSGs (Nover et al., 1983, 1989). P100
fractions from control and pre-induced cells contained putative HSG
precursors and proteasomes (Nover et al., 1989). We prepared P30s and
P100s from control, pre-induced, stressed, and recovered cells, and analyzed their proteins by SDS-PAGE, followed by Coomassie-blue staining and western blotting (Fig. 1).
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| Figure 1.
Proteins in npRNP sediments from control (c),
pre-induced (p), stressed (s), and recovered (r) tomato cells. A,
Schematic representation of heat-stress treatment and isolation of
npRNPs. B, Coomassie blue staining of proteins isolated from different
cytoplasmic RNP sediments after SDS-PAGE. C, Immunoblots of RNP
sediments probed for HSP70, small HSPs, HsfA2, the proteasome C2
subunit, and tubulin, respectively.
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Coomassie-blue staining (Fig. 1B) revealed no dramatic change in
protein patterns of P30s or P100 from cells kept at different temperatures. Pellets contained HSPs and proteasome proteins, but many
other proteins as well. Western blots revealed temperature-dependent changes in protein content (Fig. 1C). P30s and P100s from control cells
lacked small HSPs. After pre-induction, small HSPs were present in the
HSG-precursor-containing P100, which was found in P30 during stress
(presumably in HSGs) and reappeared in P100s during recovery. The lack
of small HSPs in P100s from stressed cells indicate that virtually all
small cytoplasmic HSPs reside in granules after prolonged stress, in
agreement with the cytolocalization shown in Figure 6 (see below).
Stress also resulted in a large amount of HSP70 in P30. The different
distribution of HSP70 in control cells, where it could be detected in
all fractions, and the absence of small HSPs in sediments from control
cells reflects the fact that cells synthesize constitutive and
stress-induced versions of HSP70. The mRNA-rich P30 fractions lacked
the proteasome C2 subunit, which was found in all P100s (Fig. 1C).
HsfA2, which is thought to associate with HSGs after prolonged stress
(Scharf et al., 1998), was indeed found in P30 from stressed cells. In contrast to HSP70 and the small HSPs, which were present in HSG precursors, HsfA2 associated with mature HSGs only and was not found in
any of the P100s. The cytoskeleton component tubulin was abundant in
all P100s and in P30s from stressed cells. A small amount was also
detected in P30s from pre-induced and recovered cells. The large amount
of tubulin in P30s from stressed cells coincided with a slight
reduction in the corresponding P100s.
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| Figure 6.
Localization of fluorescin-labeled histone H4 and
small Hsp mRNAs (green) and HSGs in control and stressed tobacco
protoplasts. HSGs were labeled with anti-Hsp17 and TRITC-conjugated
secondary antibodies (red). Overlays of the top and middle panels are
displayed at the bottom.
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Repressed Messengers Shuttle between Polysomes and Nonpolysomal
RNPs
In vitro translation of mRNAs from polysomes and npRNPs from
tomato cells suggests that most translatable housekeeping messengers leave the polysomes upon heat stress and are replaced by HSP
transcripts (Nover et al., 1989). However, the poly(A)-tail length of
HSP mRNAs varied with temperature (Osteryoung et al., 1993; Dellavalle et al., 1994), and repressed mRNAs on polysomes and heat-stress messengers in npRNPs could escape detection if their translatability was affected by poly(A)-tail shortening or other modifications. To
detect mRNAs independent of their translatability, we analyzed mRNA
distribution by RNA blotting instead of indirectly assaying them by in
vitro translation; this way, we could study defined messengers as
opposed to proteins produced from a large pool of unidentified
transcripts.
Total, polysomal, and nonpolysomal RNA were isolated from cells prior
to stress and after pre-induction, heat stress, and recovery. Following
the method of Nover et al. (1989), we isolated nonpolysomal RNAs from
P30 fractions after the polysomes were disrupted with high amounts
of KCl, EDTA, and Nonidet P-40 to prevent their
cosedimentation with npRNPs. RNA from different RNP fractions was
separated on a gel and stained with ethidium bromide (Fig.
2A). The lanes with RNA from npRNPs were
deliberately overloaded. Despite the large amount of RNA applied, rRNAs
are not visible in these lanes, indicating that these fractions are indeed nonpolysomal and the messengers therein are therefore not translated.
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| Figure 2.
RNA distribution during heat stress and recovery.
Total, polysomal, and nonpolysomal RNA was isolated from control (lanes
1), pre-induced (lanes 2), stressed (lanes 3), and recovered (lanes 4)
cells separated by electrophoresis and stained with ethidium bromide
(A) or blotted and probed for the transcripts indicated (B). RNA was
normalized to the number of cells isolated, except the last four lanes
in A were deliberately overloaded to display the presence of mRNAs and
the absence of rRNAs. CaM, calmodulin; H4, histone H4; sdh, steroid
dehydrogenase; cyp, cyclophilin; sHsp, small HSP.
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For northern analysis (Fig. 2B), however, RNPs were normalized for the
number of cells to avoid loading artifacts, because the amount of mRNA
in npRNPs varied with temperature and was markedly increased by stress
(not shown). Additionally, normalization for RNA content would result
in overrepresentation of messengers from control npRNPs because RNA is
almost absent in these particles. We propose that mRNAs detected in
nonpolysomal populations indeed reflect inactive transcripts, a
conclusion supported by the fact that P30 fractions from stressed cells
contained much more RNA than other P30s, in accord with the effects of
translational repression during heat stress.
Stress and recovery had no detectable influence on the total amount of
mRNAs for calmodulin, histone H4, steroid dehydrogenase, and
cyclophilin (Fig. 2B). HsfA2 and small HSP (HSP17.7) transcripts were
not found in control cells, but appeared upon pre-induction. Cyclophilin, small HSP, and HsfA2 messengers were translated during heat stress. Figure 2B shows that these transcripts were indeed present
mainly in polysomes. However, HsfA2 mRNA appeared in npRNPs after
prolonged stress. Together with the appearance of the corresponding protein in cytoplasmic aggregates (Scharf et al., 1998; Fig. 1C), this
result suggests that HsfA2 was active in the early heat-stress response
but was repressed when stress continued. Moreover, appearance of this
stress-induced transcript proves that the mRNAs in our npRNPs from
stressed cells represented messengers inactivated by stress and did not
merely reflect aggregation of pre-existing mRNPs.
Some small HSP RNA was detected in npRNPs after large numbers of
small HSPs were produced, but most small HSP messengers remained on
polysomes and the synthesis of small HSPs continued during the entire
stress phase and far into the recovery period. Histone H4, calmodulin,
and steroid dehydrogenase were not stress induced, because their
messengers were found in npRNPs during heat stress and reappeared in
polysomes during recovery. In agreement with the observation that
translation of non-HSP proteins resumed upon recovery from stress when
de novo synthesis of mRNA was blocked by actinomycin D (Nover and
Scharf, 1984), our data show that a set of selected
heat-stress-repressed messengers is stored in npRNPs in tomato cells
during stress.
Isolated Storage RNPs Bound mRNAs in a Nuclease-Resistant Form
Independent of Transcript Type
Selective mRNA distribution over polysomes and npRNPs may
be caused by a combination of the translational apparatus, the storage machinery, and/or other factors. To study the possible role of storage
RNPs in this process, we tested their capacity to bind mRNAs in vitro.
NpRNPs from control, pre-induced, stressed, and recovered cells were
incubated with transcripts encoding tomato histone H4 and HSP17.7, the
former being repressed and the latter induced upon stress. We also
tested firefly luciferase messengers with or without the poly(A) tail
for interaction with tomato RNPs. These luciferase constructs were
translated when introduced into plant cells, but were repressed and
stabilized by heat stress (Gallie et al., 1995).
The npRNPs in the P30s and P100s protected messengers from degradation
by micrococcal nuclease (shown for histone H4 mRNA in Fig.
3A), regardless of the transcript (shown
for the P30 from stressed cells in Fig. 3B) . All other combinations of
npRNPs and mRNAs yielded the same result (not shown). Messengers
incubated with BSA but without nuclease remained intact (Fig. 3). BSA
itself did not protect the transcript, and neither did the P30 from
stressed cells when it was heated to 95°C for 5 min before the
addition of RNA (Fig. 3A), indicating that the mRNAs were protected
from the nuclease by proteins in the npRNP preparations. The poly(A) tail was not involved in this protection, because messengers with and
without the tail survived the nuclease treatment (Fig. 3B).
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| Figure 3.
Protection of mRNAs from nuclease digestion by
npRNPs. RNA survival was monitored after electrophoresis. A, H4 mRNA
incubated with BSA (lanes 1 and 2), P30s (lanes 30) and P100s (lanes
100) from control (ctrl), pre-induced (pre), heat-stressed (hs), and
recovered (rec) cells, and with heat-denatured P30 from stressed cells
(lane 3). No nuclease was added to the samples in lane 1. B, Messengers
for histone H4 , HSP17.7 (sHsp), and luciferase with
(lucA50) and without Luc the poly(A+) tail
incubated with BSA (lanes 1 and 2) and P30s from stressed cells (lanes
3).
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Transcripts with or without a methylated GpppG cap were protected
equally efficiently (not shown). The same RNAs were retarded on a
nondenaturing gel after incubation with npRNPs but not when incubated
with BSA, as shown in Figure 4 for
histone H4 mRNA with P30 from stressed cells and P100s from control,
pre-induced, and stressed cells. We also observed insensitivity to
nuclease and a shift in the elution peak upon gel filtration (see
below) when mRNAs were incubated with npRNPs in a buffer
containing high amounts of salt and detergent (250 mM NaCl, 0.5% Nonidet P-40, and 0.2% sodium-lauroyl-sarcosine), which suggests a specific interaction. In
the nuclease and gel-retardation assays and in the gel-filtration experiment, the RNPs did not distinguish stress-induced from repressed messengers, suggesting that selection in vivo requires other factors.
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| Figure 4.
Gel retardation of mRNA by npRNPs. Radiolabeled
histone H4 mRNA (lane 1) and the same RNA incubated with BSA (lane 2),
P100s from control (lane 3), pre-induced (lane 4), and stressed cells
(lane 5), and P30 from stressed cells (lane 6) was separated on a
nondenaturing gel.
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mRNA Binding by Fractions of RNP Sediments
The idea that HSGs or proteasomes are associated with mRNPs is
based on cosedimentation after ultracentrifugation. However, sediments
did not only contain proteasomes, HSGs, and their precursors, but
many other proteins as well. Unfortunately, the large aggregates in
short-spin sediments (P30s) resisted chromatographic separation. Proteasomes and HSG precursors in P100s were separated by
chromatography, but the RNA content of the resulting fractions was not
determined (Nover et al., 1989). Detection of endogenous messengers in
the P100s was not feasible as the lengthy isolation procedure yielded RNA that was not suitable for northern analysis. Therefore, we again
fractionated the sedimented RNPs by gel filtration after mRNA binding
in vitro. For this purpose, RNP fractions were incubated with
radiolabeled mRNA and separated on a Superdex S200 column.
P100s from control and pre-induced cells eluted as complexes of
distinct protein composition, as shown in Figure
5 for P100s from pre-induced cells
incubated with histone H4 mRNA. Elution of labeled messenger was
shifted (Fig. 5A) to fractions with major protein bands at
approximately 40, 55, 60, 70, and >100 kD and minor bands in the 25- to 35-kD range (Fig. 5B). This shift was also observed using mRNAs
coding for small HSPs and luciferase (not shown), but elution of UTP
remained unaltered (Fig. 5A). HSP70 and small HSPs eluted in a broad
range of fractions, reflecting the size heterogeneity of small HSP
oligomers and (pre)HSGs. The peak fractions did not coincide with those
of the reporter RNA (Fig. 5C). This result is in agreement with
observations that these HSPs were not required for mRNA binding (Gallie
and Pitto, 1996; Figs. 3 and 4) and with our finding that elution of
all messengers was shifted when incubated with npRNPs from control cells, which do not contain small HSPs (not shown).
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| Figure 5.
Gel-filtration fractionation of npRNP sediments.
A, Elution of [-32P]UTP and npRNPs (P100) from
pre-induced tomato cells (top) and radiolabeled histone H4 mRNA in the
absence (middle) and presence (bottom) of npRNPs. Bars, eluted
radioactivity as percentage of total counts applied. Arrowhead, Elution
of UTP in the absence of npRNPs. Solid line,
A280 in arbitrary units. Numbers below the
graphs mark fractions of 10 mL. B, Fractions separated by SDS-PAGE and
stained with Coomassie blue. Lane m, Marker. C, Immunoblots of
fractions using antisera for Hsp70 and small Hsp (sHsp). Numbers in B
and C correspond to the fraction numbers in A. Lanes c, Control
(nonpolysomal sediment before gel filtration).
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Localization of Fluorescein-Labeled mRNAs in Tobacco
Protoplasts
In our in vitro RNA-binding experiments, mRNPs did not distinguish
stress-induced from repressed messengers. To study mRNA during stress
within cells, we introduced fluorescent transcripts into protoplasts
and studied their localization by fluorescence microscopy. Since our
tomato cell culture resisted transformation (not shown) we used tobacco
leaf protoplasts, which are able to translate synthetic mRNAs (Gallie
et al., 1991). UV crosslinking of radioactive messengers to proteins in
intact tobacco protoplasts and lysed tomato cells labeled three major
bands at approximately 30, 55, and >200 kD (R. Stuger, unpublished
data), which is similar to the labeling pattern seen after the
cross-linking of transcripts injected into Xenopus laevis
oocytes by Meric et al. (1997).
After insertion of RNAs encoding histone H4 and HSP17.7, protoplasts
were heat stressed or kept at 25°C for 2 h. This amount of time
was necessary to form HSGs. Analysis of radiolabeled mRNAs re-isolated
after introduction into tobacco protoplasts indicated that
approximately one-half of the RNA remained intact after 2 h (not
shown), comparable to the half-life of approximately 100 min for capped
luciferase mRNA in tobacco protoplasts (Gallie et al., 1991). Such
luciferase mRNA could still be translated into active luciferase after
this time. The HSGs were detected with an antibody against small HSPs.
HSGs were visible in most of the heat-stressed cells. In some cells the
amount of RNA was below the detection limit, and in others the amount
was too high to draw conclusions on the subcellular localization. The
majority of cells contained intermediate amounts of RNA. Figure
6 shows RNAs and small HSPs as they
appeared in most of the cells. The messengers for histone H4 and
HSP17.7 resided in the cytoplasm but did not colocalize with HSGs,
although a little overlap was sometimes visible. We obtained the same
result with luciferase transcripts with and without a poly(A) tail:
Neither of the tested reporter mRNAs colocalized with the small HSPs
(not shown).
| |
DISCUSSION |
Transfer of heat-stress-repressed mRNAs from polysomes to
cytoplasmic nonpolysomal storage particles in tomato cells and
relocation to polysomes during recovery illustrates the diversity of
selective mRNA repression during stress in different organisms. Whereas the investigated non-heat-stress transcripts were driven to npRNPs upon
heat stress, stress-induced messengers were associated mainly with
polysomes. Messengers for HsfA2, although present in polysomes early in
the stress response, entered npRNPs after prolonged stress. This
relocation coincided with the clustering of HsfA2 in cytoplasmic HSGs,
where it was obviously not active as a transcriptional activator. The
increased amount of mRNA and aggregated protein in sediments from
stressed cells and the fact that cells also keep transcripts in npRNPs
under nonstress conditions raise the possibility that the mRNAs in the
P30s from stressed cells do not represent messengers silenced during
stress but instead reflect the aggregation of npRNPs present before
stress. However, the presence of the stress-induced HsfA2 mRNA on
polysomes after pre-induction and its appearance in npRNPs during
stress shows that the messengers in the P30s did indeed represent mRNAs
dissociated from polysomes as a consequence of stress.
Our data and previous findings (Nover and Scharf, 1984) suggest that
non-heat-stress-induced transcripts shuttle from polysomes to npRNPs
and back. Similar shuttling was proposed for somatic carrot embryos,
whereas small HSP RNA was not found on polysomes in
heat-stressed carrot suspension-cultured cells (Apuja and Zimmerman, 1992). However, small HSP mRNA was abundant in polysomes from tomato suspension-cultured cells. Discrimination between different transcripts was not seen when mRNAs were added to isolated
npRNPs. All npRNPs bound non-heat-stress mRNA and heat-stress mRNA,
although the latter were mainly polysome-associated in vivo. We propose that the selection mechanism must reside elsewhere.
The binding of transcripts to small HSP-free RNPs together with
different cytoplasmic localization of HSGs and mRNAs are in agreement with the finding that repression and stabilization of non-heat-stress transcripts does not require HSPs or heat-stress mRNAs
(Gallie and Pitto, 1996). We cannot rule out the possibility that
RNA-binding proteins (partially) unfolded by stress are captured by
HSGs, an interaction that may be expected because small HSPs and other
molecular chaperones are abundant in HSGs (Nover et al., 1983; Arrigo,
1987; Helm et al., 1997; Jinn et al., 1997). Although we found no
interaction between messengers and small HSPs in vitro or in tobacco
protoplasts, it remains possible that small HSPs are involved in mRNA
handling during recovery from stress.
The binding of mRNAs to C2 proteasome subunit-free RNP preparations is
in contradiction to the proposed role of proteasomes in mRNA
repression, although proteasomes from sea urchin (Akhayat et al.,
1987), duck, mouse, and HeLa cells (Schmid et al., 1984) were found to
cosediment with mRNPs. The observation that X. laevis proteasomes form granular clusters that colocalize with actin and
myosin (Ryabova et al., 1994) and that proteasomes from mammalian cells
also interact with the cytoskeleton (Olink-Coux et al., 1994) may
explain cosedimentation, because nonpolysomal mRNPs (Scherrer and Bey,
1994), polysomes (Hesketh, 1994), and possibly mRNA itself (Muench et
al., 1998) also associate with the cytoskeleton.
Kraemer and Blobel (1997) found mRNPs appearing as coarse aggregates in
HeLa cells treated with cytoskeleton-disrupting agents. RNPs and
proteasomes, separated because of their association with different
sections of the cytoskeleton, may come together when this structure
collapses. We found that the HSG- and mRNA-rich P30s from stressed
cells contained tubulin, whereas P100s from stressed cells contained
less tubulin than the other P100s. Although P30s from stressed cells
carried a large amount of tubulin, the proteasome C2 subunit was not
present. However, the possibility remains that proteasomes without
the C2 subunit interact with RNPs in a biologically
significant way.
Translation-initiation factors and the 72-kD poly(A)- binding protein
represent the only identified and characterized plant cytoplasmic
mRNA-binding proteins (for review, see Albá and Pagés, 1998). The UV-crosslinking of radiolabeled mRNA to proteins in tomato
cell lysate and tobacco protoplasts yielded a labeling pattern similar
to the one found using X. laevis oocytes, with an intensely
labeled band at about 55 kD. The X. laevis protein was
identified as core mRNP FRGY2, which is abundant in repressed mRNPs
(Meric et al., 1997). Whether core mRNPs and poly(A)-binding proteins
are present in npRNPs from tomato awaits further investigation. The
type of mechanism that targets mRNAs to storage particles remains
unknown. The competition between the translation machinery and npRNPs
may separate translation-competent transcripts from inactive mRNAs.
Targeting of messengers to storage RNPs and the subsequent recruitment
of HSP mRNAs for translation is also possible. It will be easier
to investigate these options when we know more about cytoplasmic
mRNA-binding proteins in plants.
| |
FOOTNOTES |
1
This work was funded by the Deutsche
Forschungsgemeinschaft (grant no. No 249/1).
2
Present address: Department of Molecular Cell
Physiology, Free University, de Boelelaan 1087, 1081 HV Amsterdam, The
Netherlands.
3
Present address: Genetics Unit, Novartis
Forschungsinstitut, Brunnerstrasse 59, 1235 Wien, Austria.
*
Corresponding author; e-mail
forreiter{at}cellbiology.unifrankfurt.de; fax 49-69-798-29286.
Received September 21, 1998;
accepted January 31, 1999.
| |
ABBREVIATIONS |
Abbreviations:
Hsf, heat-stress transcription factor.
HSG, heat-stress granule.
HSP, heat-stress protein.
npRNP, nonpolysomal RNP.
RNP, ribonucleoprotein.
| |
ACKNOWLEDGMENTS |
We thank Daniel Gallie for the generous gift of pT7-LUC-A50;
Martin Ganal for the steroid dehydrogenase clone; Masaaki Umeda for
sharing the proteasome antibody; Michael Hoff and Daniela Löw for
subcloning the small HSP cDNAs; Lutz Nover, Rob Benne, Dave Speijer,
and Daniela Löw for helpful comments on the manuscript; and all
members of the Molecular Cell Biology Department of Goethe University
for sharing clones, antibodies, and ideas.
| |
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Abstract
Introduction